Project Summary Our goal is to characterize immune signatures (IMS) of T cell subsets associated with protection vs. susceptibility to severe disease following infection with dengue virus, and IMS associated with administration of DENV experimental vaccines. To accomplish this goal we will analyze CD4+ and CD8+ memory T cells in the context of natural endemic exposure to DENV, DENV disease and vaccination. To ensure generality of the findings, samples from two rather distinct endemic areas (Nicaragua and Sri Lanka) will be analyzed. In Aim 1 we will compare the IMS of total CD4+ and CD8+ memory T cell populations in infected/immune individuals to those of non-infected individuals from the same population. These IMS will be compared with those obtained from specific subsets of CD4+ and CD8+ T cells, identified on the basis of memory markers and chemokine receptors, and implicated in protection from severe disease. We will further compare these IMS with those of DENV antigen specific T cells derived from the same phenotypic subsets, and restricted by HLA class I and class II molecules associated with decreased or increased susceptibility to severe disease. In Aim 2 we will study T cell responses associated with severe disease, defined by T cell phenotypes observed in longitudinal sampling of PBMCs from DENV infected individuals associated with differential disease severity, from early infection time points into convalescence. Finally, in Aim 3 the phenotypes defined above will be compared with experimental DENV live-attenuated vaccines (DLAV) and those observed following vaccination against another flavivirus (yellow fever; YF). The IMS associated with specific T cell subsets will be compared with IMS associated with DENV-specific T cells, isolated on the basis of tetramer staining techniques. We will utilize a variety of complementary approaches including: cytometry analysis of a large panel of phenotypic markers, RNA-Seq profiling and single cell analysis, mesoscale assays of cytokine production and proteomic analysis of specific memory T cell subsets. These results will be further correlated with tertiary outcome measurements such as viral titers, antibody titers, virus neutralization assays, clinical outcomes, HLA type and other individual donor-related characteristics. Our results will be compared with those obtained by Project 2, which studies a chronic bacterial disease (Tuberculosis) in the context of natural exposure, disease and vaccination. These results will also be the basis of initial validation for additional RNAi and epigenetic profiling performed in Project 3.